Nucleus export reporter system

ABSTRACT

The present invention relates to reporter systems for RNA export, methods for searching for molecules which influence RNA export, and a method, based on these methods, for detecting a viral infection.

The present invention relates to reporter systems which permit theidentification of proteins, signal sequences and/or active substancecandidates which regulate the export of RNAs (ribonucleic acids) and, inparticular, viral RNAs from the cell nucleus of eukaryotic cells.

A wide variety of viruses depend on active export of their incompletelyspliced transcripts from the cell nucleus of the infected host cell.This can either take place by use of an RNA signal in cis within theviral transcripts (constitutive transport elements), or takes place withthe aid of viral proteins. Cis-active transport elements are used forexample from MPMV-CTE (Mason-Pfizer monkey virus constitutive transportelement), SRV-CTE (simian retrovirus constitutive transport element),hepatitis B virus PRE (posttranscriptional regulatory element) and HSV(herpes simplex virus) (within the TK (thymidine kinase) gene). TheseRNA elements recruit cellular factors and export pathways in order toenable nuclear export of the viral transcripts. An alternativepossibility is for nuclear export also to be mediated by an exportfactor which specifically binds to a target sequence within the viraltranscripts and transports the latter into the cytoplasm throughinteraction with cellular factors. Thus, for example, Ad-5 transcriptsare exported with the aid of the 34K and E4orf6 proteins, EBV(Epstein-Barr virus) transcripts with the aid of the EB2 protein,herpes-virus saimiri transcripts with the aid of the ORF 57 geneproduct, HSV (herpes simplex virus) transcripts with the aid of the ICP27 protein, HTLV-I and II (human T-cell leukemia virus I and II)transcripts with the aid of the Rex proteins, EIAV (equine infectiousanemia virus), SIV (simian immunodeficiency virus) and HIV-1 and HIV-2(human immunodeficiency virus 1 and 2) transcripts with the aid of theRev proteins.

Nuclear export which has been investigated best is that of late HIV-1transcripts mediated by HIV-1 Rev. Like all lentiviruses HIV-1 dependson a plurality of genes being activated from only one proviral templateand being expressed in a fixed time sequence. Different genes aregenerated from a primary transcript which is only ˜9 kB in size byalternative splicing events and other regulatory mechanisms taking placeat the RNA level. These viral transcripts can be divided into 3 classeson the basis of their size: ˜9 kB unspliced (gag, pol), ˜4 kB singlyspliced (env, vif, vpr, vpu) and ˜2 kB multiply spliced (rev, tat, nef)RNAs.

Besides the occurrence of incompletely to multiply spliced transcripts,it is additionally possible to observe a time sequence in the expressionof these different RNA species. Thus, only the multiply spliced ˜2 kBRNAs, and their gene products Rev, Tat and Nef, are detectable in theearly phase of replication in the cytoplasm of the infected cells. Onlyafter a time lag do the unspliced (˜9 kB) and singly (˜4 kB) splicedtranscripts and their gene products Gag, Pol and Env then also appeartherein. However, the singly and unspliced transcripts can never bedetected in the cytoplasm of cells infected with viral mutants lackingan active Rev protein. The unspliced and singly spliced transcripts thenaccumulate in the nucleus, and the late structural proteins (Gag, Env)and enzymes (Pol) translated from them cannot be formed. The viral Revprotein is thus essentially involved in the time-regulated expression ofthe viral genes.

HIV-1 Rev, just like the RNA transport molecules mentioned above, areshuttle proteins which transport viral RNAs via the interaction with anRNA target sequence located within viral transcripts the latter out ofthe nucleus into the cytoplasm. Thus, HIV-1 Rev binds specifically inthe nucleus to its RNA target structure RRE, the Rev-responsive element(see FIG. 5 A/B). This region, which is 351 nucleotides (Nt) long, islocated within the Env reading frame and is thus a constituent of allunspliced and singly spliced transcripts. This ribonucleoprotein (RNP)complex is subsequently exported out of the cell nucleus via interactionwith cellular factors. A leucine-rich sequence located at the C terminusis necessary for this and, as nuclear export sequence (NES), mediatesnuclear translocation of the Rev protein through use of cellularmechanisms (Pollard and Malim, 1998).

The reason why the late transcripts remain in the nucleus in the absenceof Rev, which is a necessary condition for the Rev-dependence and thusthe time-regulated expression of Gag, Pol and Env, is stillcontroversial. In principle there are two main alternative ideas aboutthe nuclear retention of late transcripts.

It is assumed that a cellular transcript can leave the cell nucleus onlywhen the splicing process is entirely complete, or all active splicesites have been deleted from the primary transcript. The late viraltranscripts are intron-containing, only incompletely spliced pre-mRNAswhich are transported into the cytoplasm with the aid of Rev and RRE.The influence of the cellular splicing machinery on the nuclearretention of the late transcripts was therefore investigated at an earlydate (Mikaelian et al., 1996; Kjems et al., 1991; Kjems and Sharp, 1993;Chang and Sharp, 1989; Powell et al., 1997; Lu et al., 1990; O'Reilly etal., 1995). The presence of splice sites differing in activity appearsto make the splicing process only suboptimal in the case of HIV-1transcripts. Several groups have therefore hypothesized that Rev makesit possible for transcripts which are retained within the splicingmachinery through the formation of inefficient splicing complexes to beexported.

However, contrary to this it has been possible to show that expressionof the late HIV-1 genes such as, for example, Env remains repressed evenin the absence of active splice sites, and thus the influence of thesplicing machinery appears to be more indirect (Nasioulas et al., 1994).This is why so-called inhibitory sequences (INS) or cis-active repressorelements (CRS) within the reading frames, which adversely influenceexpression, have been postulated (Nasioulas et al., 1994; Olsen et al.,1992; Schwartz et al., 1992b; Maldarelli et al., 1991). However, theserepressor sequences which are located inside the coding mRNA have nocommon sequence motif like, for example, the AUUUA instability motifinside the 3′-UTR of the unstable GM-CSF mRNA (Chen and Shyu, 1995), butare conspicuous only by their high A/U content throughout. Thus, fusionof the postulated INS-containing fragments from reading frames of lategenes (such as Gag and Env) to a CAT reporter system resulted in areduced reporter activity (Cochrane et al., 1991; Rosen et al., 1988;Schwartz et al., 1992b). It was possible to abolish again in part thisreduction in the expression of Gag and Pol in part by multiple silentpoint mutations within the wobble positions (Schwartz et al., 1992a;Schneider et al., 1997). The unspliced and singly spliced HIV-1 mRNAsthus appear to have cis-active repressor elements which are eitherdeleted by multiple splicing or overcome by a Rev/RRE-mediated nuclearexport.

There is great medical interest in molecules which modulate, inparticular inhibit, the export of RNA, in particular viral RNA, from thecell nucleus. For example, HIV-1 Rev is an essential factor duringreplication of HI viruses both in cell culture and in vivo. (Feinberg,Jarrett, 1986; Iversen, Shpaer, 1995; Sodroski, Goh, 1986). This makesRev an attractive target for therapeutic agents with antiviral activity.

Rev-sensitive reporter systems can be used for testing active substanceswhich suppress a Rev function and thus prevent productive replication ofHIV-1. A wide variety of systems has been used to date for evaluatingthe Rev-dependent HIV-1 gene expression. These extend from mutatedprovirus constructs (Borg et al., 1997; Malim and Cullen, 1993) viachimeric Rev-sensitive β-globin genes (Chang and Sharp, 1989; Mikaelianet al., 1996) to subgenetic fragments fused to reporter genes (Schwartzet al., 1992a; Schwartz et al., 1992b).

However, the systems used to date are not suitable for thehigh-throughput testing (HTT) of therapeutic agents with aRev-inhibiting effect, or for the isolation of specific inhibitors ofthe Rev function from a randomized gene library: working with viralsystems or provirus constructs capable of replication requires S3 safetylaboratories, and elaborate and costly (p24 capture ELISA) detectionmethods which do not permit HTT per se or make it unattractive. ComplexRev-dependent β-globin reporter systems are suitable only for basicresearch, and require special, demanding laboratory methods (Northernblot, RNA protection assay), which likewise preclude HTT. Reportersystems based on a specific enzymatic reaction for detectingRev-sensitive gene expression permit HTT at least in some cases. Thus,it has been possible to convert expression of the chloramphenicolacetyltransferase (CAT) gene into Rev dependence by placing the CATreading frame together with the RRE region inside an intron sequence(Luo et al., 1994; Iacampo, Cochrane, 1996; Hope, Huang, 1990), Thisreporter construct (pDM128 and derivatives) has a number ofdisadvantages, however:

-   (a) The assay cannot be used on a single cell basis. Thus cells    cannot be separated by FACS but must necessarily be lysed for    analysis.-   (b) Simple, easily automatable, optical testing of a Rev action is    not possible.-   (c) Unspliced CAT RNAs can be detected in the cytoplasm of cells    transfected with pDM128 even in the absence of Rev, i.e. a Rev    dependence is incorrectly simulated in the assay even in the absence    of Rev.-   (d) The presence of an intact cellular splicing machinery is a    precondition for the assay. Molecules which influence splicing    processes are incorrectly classified as modulators of RNA export in    the assay.

It was therefore an object of the present invention to provide a methodfor detecting RNA export from the cell nucleus of a eukaryotic cellwhich does not have the abovementioned disadvantages of the prior art.

This object is achieved by a method comprising the steps:

-   (a) provision of a nucleic acid which codes for a reporter protein    and whose transcript is exported from the cell nucleus depending on    the presence    -   (i) of a cis-active RNA export signal in operative linkage with        the nucleic acid and    -   (ii) of a trans-active factor and, where appropriate,    -   (iii) of a functional 5′ splice donor in the absence of a        functional 3′ splice acceptor,-   (b) introduction of the nucleic acid into the cell nucleus of the    target cell so that it is present therein in operative linkage with    a transcription control sequence, and that on transcription of the    nucleic acid there is generation of a transcript in which the    segment of the transcript coding for the reporter protein cannot be    subjected to any splicing process,-   (c) transcription of the nucleic acid and-   (d) determination of whether the resulting transcript is exported    from the cell nucleus.

The presence of the 5′ splice donor must not lead to a splicing eventbeing possible as in the case of the constructs of Lu et al.Accordingly, the 5′ splice donor must not be confronted by a 3′ spliceacceptor or at least any efficiently utilized splice acceptor.

The cis-active RNA export signal may be a previously known RNA exportsignal. In this case, the RNA export signal is preferably positioned inrelation to the reporter gene in analogy to the natural occurrence ofthe export signal. Thus, for example, in the case of the Rev-responsiveelement of HIV-1 the signal is cloned downstream of the reporter gene.Conversely, the export signals of adenoviruses are preferably disposedupstream of the reporter gene.

The method of the invention is suitable for the identification ofunknown RNA export signals. For this purpose, the reporter gene isoperatively linked in the nucleic acid to a library of gene fragments,and constructs which make RNA export possible are sought. The genefragment library preferably comprises genes or gene fragments from donororganisms or viruses known to have efficient RNA export signals. If itis intended specifically to search for RNA export signals which requirethe presence of viral trans-acting factors, these must likewise beprovided in the cell.

It is conversely possible, if a cis-active export signal is known and atrans-active factor is to be identified, to use a construct as describedabove to search for such a factor. In this case, the nucleic acidconstruct comprises both the reporter gene and the cis-active signal. Itis then possible to use the reporter system described above to search inexpression libraries for trans-active factors. The trans-active factorsare preferably viral adaptor molecules which mediate an interaction withthe cellular RNA export machinery, but they may also be cellularmolecules. Such a trans-active factor includes for the purposes of thisapplication both a single protein and a protein complex.

If all the cis- and trans-acting factors necessary for RNA export areprovided, the system is suitable for identifying molecules whichinfluence RNA export. The molecule which influences RNA export may be,for example,

-   (a) a small molecule, typically having a molecular mass of less than    2 000 Da,-   (b) DNA or RNA, derivatives or mimetics thereof, which act at the    nucleic acid level or interact with proteins involved in RNA export,    or-   (c) a peptide, a modified peptide, protein or modified protein which    interacts with nucleic acids or interacts with proteins involved in    RNA export.

An example of a small molecule as in (a) is leptomycin B, a knowninhibitor of Rev activity. An example of a nucleic acid as in (b) arethe known RRE decoys and an RNA intramer. An example of a protein as in(c) is the transdominant-negative Rev mutant RevM10. It is possible inparticular for the nucleic acids to be derived from a gene library orfor the proteins to be gene products of the genes of a gene library. Inthese cases, the advantage of the system of the invention, of dispensingwith lysis or fixation of the cells, is very particularly evident.

The activity of leptomycin B and of the transdominant-negative Revmutant RevM10 were detectable with the reporter system of the invention(with hivGFP as reporter protein). The produced Rev-sensitive reportersystem is thus suitable for detecting inhibition of viral geneexpression and thus as reporter system for identifying therapeuticagents with antiviral activity and acting on viral nuclear export.

A further substantial improvement of the reporter system of theinvention compared with the previously disclosed CAT system is theavoidance of splicing processes. In the CAT system, RNA is exported intothe cytoplasm in every case. Only in the presence of a trans-activefactor such as, for example, Rev does the latter also contain an intronon which the reporter gene is encoded. By contrast, in the reportersystem of the invention there is no need for a splicing event to takeplace. In the absence of cis- or/and trans-active signals, the RNA isjust not exported into the cytoplasm in amounts which allow theaccumulation of detectable amounts of RNA.

This can be achieved, for example, by RNA-destabilizing sequence motifssuch as, for example, AUUUA which occurs in the RNA for GM-CSF, orpreferably by a choice of codons which reduces the metabolic stabilityof the RNA. Constructs suitable for reporter systems of the inventioncan be generated for example by choosing the codon distribution likethat occurring in viral exported RNA. A choice of codons to be usedpreferably in this connection is one like that used least frequently orsecond-least frequently in mammalian cells (Ausubel et al., 1994), evenmore preferably the choice of codons is adapted to that of late HIV-1genes, and even more preferably table 1 is used for producing theRev-dependent reading frame.

For example, the choice of codons of the constitutively expressing genefor green fluorescent protein (GFP) was adapted to the choice of codonslike that to be found in late HIV-1 genes. For this purpose, the aminoacid sequence of the GFP gene product was back-translated into asynthetic GFP-encoding reading frame using the HIV-1 Gag choice ofcodons. This reading frame, called hivGFP, was then constructed as fullysynthetic reading frame using long oligonucleotides and a stepwise PCR.In addition, the authentic 5′-UTR of the Gag reading frame was put infront of the hivGFP reading frame, and the RRE was attached 3′ andcloned into an expression vector. The produced hivGFP vector proved tobe completely dependent on the presence of the Rev protein in theexpression of the autofluorescent GFP. In the absence of the 5′-UTR, RREor Rev it was not possible to detect any expression of the greenfluorescent reporter. The initial GFP gene, which was adapted in itschoice of codons to mammalian genes (huGFP), by contrast proved to beindependent of Rev, RRE or the 5′-UTR in its expression.

However, instead of adapting the choice of codons as accurately aspossible to the choice of codons of late HIV genes, it is also possiblemerely to increase the AT content. An AT content of >50% is preferablyaimed at. Increasing the AT content or adapting the codon usagepreferably takes place by silent mutations or by mutations which do notdestroy the activity of the reporter protein. The choice of codons neednot be adapted if the A/T content of said gene is already more than 50%.Genes with a codon usage differing from the wild type can be produced asindicated in the example for example from long oligonucleotides and witha stepwise PCR.

The reporter protein preferably used is a fluorescent protein, becauseof the particularly simple readability and the suitability forhigh-throughput tests. Examples of autofluorescent reporter proteins arethe green fluorescent protein GFP, the blue fluorescent protein BFP, thered fluorescent protein RFP, the yellow fluorescent protein YFP, orderivatives of these autofluorescent proteins which display increasedfluorescence, such as the enhanced green fluorescent protein eGFP, theenhanced blue fluorescent protein eBFP, the enhanced red fluorescentprotein eRFP or the enhanced yellow fluorescent protein eYFP (Clontech).

Instead of using fluorescent proteins it is also possible to use otherproteins as long as their activity is easily detectable. Examples arethe gene for luciferase LUC, the gene for alkaline phosphatase AP, thegene for secretory alkaline phosphatase SEAP or the gene forchoramphenicol acetyltransferase CAT.

Immunologically detectable proteins are likewise suitable as reporterproteins. It is sufficient for rapid immunological detection of the geneproduct, of parts of the gene product or of epitopes to be possible. Afrequently used example is the influenzae Flag-tag.

Proteins capable of positive or negative selection are also suitable asreporter proteins. For example, the neomycin-resistance gene prevents atranslation block caused by G418 and thus death of the cell. In HAT(hypoxanthine, aminopterin, thymidine) medium, in which de novo purineand pyrimidine biosynthesis is blocked, the activity of thymidine kinaseis essential. Conversely, it is possible to select for cells deficientin thymidine kinase by propagating the cells in bromodeoxyuridine.Likewise, the enzymic activity of the herpes viral thymidine kinase (TK)brings about the death of TK-expressing cells in the presence ofacyclovir. Further examples of markers capable of positive and negativeselection are adenine phosphoribosyl transferase (APRT),hypoxanthine-guanine phosphoribosyl transferase (HGPRT), anddihydrofolate reductase (DHFR). Azaserine is used for positive, and8-azaguanine for negative, selection for APRT and HGPRT. In the case ofDHFR, methotrexate is used for selection for the marker, and [³H]dUrd isused for selection against the marker. All the marker systems mentionedare described in detail by Kaufmann (Kaufmann, 1979).

Finally, the reporter gene may be a regulatory gene which, after itsexpression in a cell as molecular switching molecule, switches theexpression of other genes on or off. An example of such a regulatorygene which can be used is a transcription factor.

In all methods in which it is not intended to search for a cis-activeRNA export signal, the nucleic acid construct already contains acis-active RNA export element.

This may be may be a so-called constitutive transport element. No viralproteins are necessary for the export for the nuclear export of RNAswhich harbor such a constitutive transport element; the virus merelyutilizes cellular mechanisms which are already present. Examples of suchcis-active RNA export elements are MPMV CRE, RSV CTE or SRV CTE.

Many viral cis-active RNA export elements are, however, dependent onviral adaptor proteins which mediate the interaction with the cellularexport machinery. In the case of HIV, the RRE (Rev responsive element)is used as cis-active RNA export element. This signal is recognized bythe viral protein Rev which contains a leucine-rich sequence ofhydrophobic amino acids which acts as nuclear export signal (NES). TheRev receptor in nuclear export is Crm1, which is also called exportin 1.Interaction of the Rev receptor with Crm1 can be impaired by leptomycinB. It is thought that the Rex/RxRRE system of HTLV-I and HTLV-IIfunctions analogously to the Rev/RRE system of HIV-1, HIV-2 and SIV.

The RNA export signal for the RNA export systems described hereinbeforeis preferably located downstream of the structural genes. However, it isalso possible to use RNA export signals from viruses in which the RNAexport signals are located upstream of the structural genes, as occursfor example in adenoviruses. In this case, the nuclear export sequenceis preferably located upstream of the reporter gene in the RNAconstructs too.

The invention further relates to a DNA sequence which codes for areporter protein and is operatively linked to a cis-active RNA exportelement and, where appropriate, to a functional 5′ splice donor in theabsence of a functional 3′ splice acceptor, where

-   (a) the DNA sequence coding for the reporter protein is modified at    the nucleic acid level compared with the wild-type sequence,-   (b) RNA export for the modified DNA sequence takes place dependent    on the cis-active RNA export element, and-   (c) essentially no RNA export for the wild-type DNA sequence takes    place dependent on the cis-active RNA export element.

The wild type means in this connection either the wild type in the usualsense or else a gene which is optimized for expression in thecorresponding host cell, such as, for example, “humanized GFP”. Thedependence of the modified reporter RNA on the cis-active RNA exportelement is achieved by the methods described above.

Transcription of the corresponding RNA sequence is controlled by atranscription control sequence. The transcription control sequence maycomprise a constitutive or inducible promoter. Examples of constitutivepromoters which can be used are viral promoters from CMV, SV 40 or fromadenovirus or cellular promoters such as the actin promoter or celltype-specific promoters such as the MHCII promoter. Suitable induciblepromoters are tetracycline-dependent promoters (Tet on/off system), heatshock promoters, metallothionein promoters or promoters which can beinduced by glucocorticoids, such as the promoter in mouse mammary tumorvirus (MMTV) LTR (Kaufmann, 1979). An appropriate polyadenylation signalis attached to the 3′ end of the DNA for the polyadenylation of the RNAtranscripts. The statements made above apply to the choice of thereporter protein and of the cis-active RNA export signal. It isappropriate in some circumstances to provide a trans-active factor andthe reporter construct on the same plasmid.

The present invention further relates to eukaryotic cells, morepreferably mammalian cells, most preferably human cells, which aretransformed with a DNA construct as described above, where the DNAconstruct is present in a form capable of transcription. The DNAconstruct may for example exist episomally or be stably integrated intothe chromosome. It is moreover possible for one or more copies to bepresent in the cell. If an RNA export sequence which is active only inthe presence of a viral export factor is used, it is possible bytransfection or infection of the cell with expression libraries tosearch for viral factors which interact with the RNA export sequence oract further downstream in the RNA export pathway, if such factors arenot yet known. If the RNA export activity is successfully reconstituted,it must be possible to measure an increase in the expression of theRev-dependent gene by at least 2.5-fold, preferably 5-fold, morepreferably 8-fold or more.

If the factors are already known, they are preferably made available inthe cell, usually in trans. The invention therefore relates to theproduction of stable cell lines which, besides the export constructdescribed above, also harbor the gene for an additional viral exportfactor integrated chromosomally or episomally in them. It is possible touse according to the invention any eukaryotic cell in which the factorsnecessary for RNA export are present in concentrations which correspondapproximately to the natural infection model. Except for the infectionmodel described hereinafter, it is unnecessary for the cells to bepermissive for replicative infection with the viral system to beinvestigated. Examples of eukaryotic cells which can be used are H1299cells, HeLa cells, HEK cells (human embryonic kidney cells).

The viral export factor is preferably Rev from HIV-1, HIV-2, SIV andEIAV or Rex from HTLV-I and HTLV-II, or the 34K and E4orf6 proteins fromadenoviruses, or EB2 proteins from EBV, or ORF 57 gene products fromherpes virus saimiri, or ICP 27 proteins from HSV.

The present invention further relates to the production of stable celllines which, besides the Rev-dependent gene, additionally comprise oneor more constitutively expressing genes. It is possible by determiningthe rate of expression of these genes or the amount of the relevantprotein products to distinguish whether an active substances to betested specifically affects the expression dependent on the exportfactor provided in trans, or only generally blocks cellular expression.The reporter gene whose expression is regulated by the viral exportfactor, and the constitutively expressed protein must be detectable bydifferent methods. If both proteins are fluorescent, they must thereforediffer in the wavelength of the exciting light or in the wavelength ofthe emitted light. However, it is also possible to use other enzymaticmethods or the recognition of another immunological epitope.

The DNA reporter construct and a cell which can be transfected with theDNA or a cell which is already transfected with the DNA construct can beprovided as reagent kit for investigating RNA export processes. Thereagent kit preferably also comprises leptomycin B, because it ispossible with this substance specifically to block the RNA exportpathway utilized for example by HIV Rev-dependent RNA constructs (Otero,1998).

The present invention further relates to a method for detecting a viralinfection which has taken place, more preferably a retroviral viralinfection, most preferably a lentiviral viral infection. The detectionis based on the fact that a viral export factor necessary for nuclearexport is present in infected cells but not in uninfected cells. If HIVinfection is involved, the viral RNA export factor is HIV Rev, and inthe case of HTLV infection the viral RNA export factor is HTLV Rex.

Because patients' plasma is easily available, it is appropriate todetect the infection through detecting infectious viruses in the plasma.In this embodiment, a cell which is transfected with the reporterconstruct, preferably carries the reporter construct stably in itself,particularly preferably has the reporter construct stably integratedinto the chromosome, is brought into contact with the patient's plasma.The cell is infected if viruses are present in the plasma. Thenecessary, trans-active RNA export factor is provided in this way, andRNA export can be detected.

Detection of infection is also possible even if viruses are no longerdetectable in the plasma but, nevertheless, cells are infected with thevirus. In this case the infection must originate from these cells. Analternative possibility is for the cells also be transfected with thereporter construct.

The infection can, if the reporter gene is chosen suitably, be detectedby fluorescent bioanalysis, where appropriate through theautofluorescence of the reporter gene product, preferably byspectroscopy, preferably by fluorescence microscopy, more preferably byFACS analyses. This detection can take place manually or completelyautomatically in a high-throughput testing.

An infection can be detected through detection of the reporter genedependent on the viral export factor, in particular Rev, additionallyvia the enzymatic activity of the chosen reporter gene product, such asthe phosphatase activity of SEAP, the acetyltransferase activity of theCAT gene product, the luciferase activity of the luciferase gene. Thisdetection can take place manually or completely automatically in ahigh-throughput testing.

However, the antigenic properties of a reporter protein or of one ormore epitopes of the chosen reporter protein can also be utilized.Detection then takes place by suitable immunological methods such asELISA, immunofluorescence, immunoblot, FACS of immunologically labeledcells. It is possible manually or completely automatically in ahigh-throughput testing.

It was possible to show in particular that cells transfected with theRev-sensitive hivGFP together with an infectious proviral HIV-1 clone(HX10) likewise exhibit a green fluorescent reporter gene activity. Theproduced Rev-sensitive reporter system is thus suitable for detectingHIV-1 gene expression and thus as detection of infection.

A further detection of the RNA export detection of Lu et al. is thenecessity to have to lyse or fix the cells to detect the reporterprotein or the activity of the reporter protein. The invention thereforefurther relates to a method for detecting RNA export from the cellnucleus, in which a reporter protein that can be detected without lysisor fixation of the cells is used. A fluorescent protein for example issuitable for this purpose. However, it is likewise also possible to usea suitable selection marker. The advantage of this method compared withthe prior art is the possibility

-   (a) of being able to transfect or infect the cells with a gene    library-   (b) of being able to purify the genes which bring about modulation    of the RNA export from the cell nucleus by cultivating the cells and    isolating the nucleic acid.

EXAMPLES 1. Production of a Rev-Dependent GFP Gene

The intention was to construct artificially the reading frame of thegreen fluorescent protein (Gfp) gene using a choice of codons like thatto be found in HIV-1 structural genes. For this purpose, the amino acidsequence of the Gfp gene was translated into a corresponding nucleotidesequence. This was carried out with the aid of the gcg command“backtranslate” using an appropriate matrix as described in table 1.Further cleavage sites were inserted for subcloning and for attachingfurther sequence elements within untranslated regions. An exactsequence, including the cleavage sites used, is indicated in SEQ ID No.9. The sequence produced in this way was produced as completelysynthetic gene using synthetic oligonucleotides and a previouslydescribed method {Zolotukhin, Potter, 1996}. FIG. 1 depicts a comparisonof the hivGFP (choice of codons derived from HIV structural genes) andhuGEP (choice of codons derived from mammalian genes). The GFP-encodingDNA fragment (“hivGFP”) produced in this way was placed under thetranscriptional control of the cytomegalovirus (CMV) earlypromoter/enhancer (“pc-hivGFP”) in the expression vector pcDNA3.1(+)(Stratagen, Heidelberg) using the KpnI and XhoI cleavage sites. Toproduce an analogous GFP expression plasmid whose choice of codons was,however, adapted to the human system, the coding region of the humanizedGFP gene (huGFP) was amplified from a commercially obtainable vector bymeans of a polymerase chain reaction (PCR) using the oligonucleotideshu-1 and hu-2 and cloned into the expression vector pcDNA3.1(+)(Stratagen, Heidelberg) likewise using the KpnI and XhoI cleavage sites(“pc-huGFP”).

As explained in the description, an isolated (efficiently utilizable)splice donor (SD) must be put in front of the coding region in order toachieve Rev dependence of the hivGFP reporter. The HIV-1 untranslatedregion (UTR) within the late HIV-1 transcripts contains such an SD. Thisregion was amplified by means of PCR using the oligonucleotides utr-1and utr-2 from proviral HIV-1 DNA (HX10, see (Ratner et al., 1987)) andcloned directly 5′ in front of the ATG of the GFP-encoding reading frameof the pc-huGFP and pc-hivGFP constructs using the KpnI and NcoIcleavage sites. The resulting constructs have been referred tohereinafter as “pc-UTR-huGFP” and “pc-UTR-hivGFP”, respectively.

As explained in the applications, it is necessary to attach to thecoding region an RNA target sequence in order to achieve Rev dependenceof the hivGFP reporter. This target sequence interacts at the RNA leveleither with a viral nuclear export protein (in the case of HIV-1 the Revprotein) or cellular nuclear export proteins. For this reason, the HIV-1Rev-responsive element from proviral HX10 DNA was amplified by means ofPCR using the oligonucleotides rre-1 and rre-2 and cloned 3′ behind theGFP-encoding region of the pc-huGFP, pc-UTR-huGFP, pc-hivGFP,pc-UTR-hivGFP constructs using the BamHI and XhoI cleavage sites. Theresulting constructs have been referred to hereinafter as“pc-huGFP-RRE”, “pc-UTR-huGFP-RRE”, “pc-hivGFP-RRE”,“pc-UTR-hivGFP-RRE”. In addition, the MPMV constitutive transportelement CTE from proviral MPMV DNA was amplified by means of PCR usingthe oligonucleotides cte-1 and cte-2 and cloned 3′ behind theGFP-encoding region of the pc-hivGFP, pc-UTR-hivGFP constructs using theBamHI and XhoI cleavage sites. The resulting constructs have beenreferred to hereinafter as “pc-hivGFP-CTE”, “pc-UTR-hivGFP-CTE”. All theGFP-encoding constructs produced are depicted diagrammatically in FIG.2.

For the Norhern blot analyses, additionally the RRE-encoding region wascloned in antisense orientation to the T7 promoter into the pCR-Scriptvector (Stratagene, Heidelberg). In addition, the pSP6-actin constructwas kindly made available to us by F. Schwarzmann's group (IMMH,Regensburg). To provide the viral Rev protein in trans, Prof. J. Hauber(Erlangen) kindly made available a Rev expression plasmid to us.

2. Rev-Dependent GFP Expression of the hivGFP Reporter Requires theChoice of Codons of HIV Structural Genes and the 5′-UTR/SD

All the cell culture products were from Life Technologies (Karlsruhe).All mammalian cell lines were cultivated at 37° C. and 5% CO₂. The humanlung carcinoma cell line H1299 was grown in Dulbecco's modified Eaglemedium (DMEM) with L-glutamine, D-glucose (4.5 mg/ml), sodium pyruvate,10% inactivated fetal bovine serum, penicillin (100 U/ml) andstreptomycin (100 μg/ml). The cells were subcultivated in the ratio 1:10after confluence was reached.

1.5*10⁶ cells were seeded in Petri dishes (diameter: 100 mm) and, 24 hlater, transfected by calcium phosphate coprecipitation (Graham and Eb,1973) with 30 μg of indicator plasmid and 15 μg of pc-Rev or 15 μg ofpcDNA 3.1 vector. Cells and culture supernatants were harvested 48 hafter transfection. The transfected cells were washed twice withice-cold PBS (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl),scraped off in ice-cold PBS, centrifuged at 300 g for 10 min and lysedin lysis buffer (50 mM Tris-HCl, pH 8.0, 0.5% Triton X-100 (w/v)) on icefor 30 min. Insoluble constituents of the cell lysate were removed bycentrifugation at 10 000 g and 4° C. for 30 min. The total amount ofprotein in the supernatant was determined using the Bio-Rad ProteinAssay (Bio-Rad, Munich) in accordance with the manufacturer'sinstructions.

The samples were mixed with the same volume of 2× sample buffer(Laemmli, 1970) and heated at 95° C. for 5 min. 50 μg of total proteinfrom cell lysates or half the sample batch from enriched supernatants(B.4.1) were fractionated on a 12.5% SDS/polyacrylamide gel (Laemmli,1970) electrotransferred to a nitrocellulose membrane and analyzed usingthe monoclonal p²⁴-specific antibody 13-5 (Wolf et al., 1990) anddetected by means of a secondary, HRP (horse-radish preoxidase)-coupledantibody and detected by chromogenic staining.

The reporter constructs were transiently transfected into H1299 cells.The expression achieved was analyzed in the presence and absence ofRev/RRE and of the 5′-UTR/SD. The huGFP expression could not be enhancedeither by the Rev/RRE system (FIG. 3A, lanes 3, 4), nor was itsignificantly influenced by the 5′-UTR/SD (FIG. 3A, lane 5). Nor was thecombination of 5′-UTR/SD and Rev/RRE able to convert the syntheticreading frame into Rev dependence (FIG. 3A, lane 6). However, incontrast to this, the hivGFP reporter constructs adapted to the choiceof codons of HIV-1 structural genes behaved completely differently.Thus, no GFP expression was detectable in the absence of the 5′-UTR/SD,irrespective of the presence or absence of the Rev/RRE system (FIG. 3A,lane 7, 8). Only in the presence of the 5′-UTR/SD and of the hivGFPreporter gene adapted to HIV-1 structural genes was it possible todetect Rev/RRE-dependent expression of the GFP reporter (FIG. 3A, cf.lane 9 with 10).

Rev-dependent expression based on nuclear export of viral or quasi-viraltranscripts should not be attributable by a simple stabilization of thetranscripts by the Rev/RRE interaction alone. Although the Rev-M10mutant can interact with the RNA target sequence RRE and other Revproteins (mutated as well as wild type), it cannot be exported from thenucleus due to a defect within the nuclear export signal (NES) (Stauberet al, 1995; Kubota et al, 1991). GFP expression could not be achievedin the presence of the Rev-M10 protein after transfection with theUTR-hivGFP-RRE reporter (FIG. 3B, lane 3). Rev-dependent expression ofthe hivGFP reporter is thus not attributable to stabilization of the RNAby a Rev/RRE interaction but to the subsequent nuclear export throughwild-type Rev. It was moreover possible through the UTR-hivGFP-RREreporter to achieve GFP expression in the presence of cotransfectedproviral HIV-1 DNA (HX10) (FIG. 3B, lane 2). It is thus possible todetect an HI viral infection by means of the Rev formed during viralreplication by the hivGFP reporter system. In addition, hivGFPexpression using the heterologous CTE nuclear translocation systemlikewise proved to be dependent on the presence of the 5′-UTR/SD and onthe choice of codons used (FIG. 3C). This shows that the reporter systembased on this invention is based on a superordinate nuclear retentionprinciple which operates irrespective of the nuclear export mechanism.

3. Rev-Dependent GFP Expression of the hivGFP Reporter is Based onRev-Mediated Nuclear Translocation

The experiments summarized in FIG. 3 suggest that there is Rev-mediatednuclear export of the UTR-hivGFP-RRE transcripts. In order to prove thissuggestion, the subcellular distribution of the GFP-encoding transcriptswas subjected to Northern blot analysis. GFP-encoding transcripts weredetected using an RRE-specific probe. In addition, the amount andintegrity of the β-actin RNA were detected as internal control of thequality and quantity of the RNA preparation.

For this purpose, transfected cells were detached by trypsinization andwashed 2× with ice-cold PBS. 1×10⁷ cells were partially lysed with 175μl of lysis buffer (50 mM Tris, 140 mM NaCl, 1.5 mM MgCl₂, 0.5% NP-40,pH 8.0) on ice for 5 min. The cytoplasmic fraction was separated fromthe nuclear fraction by centrifugation (300×g, 2 min) and placed on ice.The nuclei were cautiously washed with lysis buffer and againcentrifuged (300×g, 2 min). The total DNA was prepared from the nucleiand from the cytoplasmic fraction in each case using the RNeasy kit(Qiagen, Hilden). The RNA preparations were taken up in RNAase-freewater and stored at −80° C. until used further.

The following solutions were used for the Northern blot analysis:

SSPE (20x): 175.3 g of NaCl, 27.6 g of NaH₂PO₄ H₂O, 7.4 g of EDTA add 11 1 1 of H₂O (adjust to pH 7.4) MOPS (10x): 83.72 g of MOPS, 8.23 g ofNaAc, 20 ml of EDTA (0.5 M) add 1 1 of H2O (adjust to pH 7.0) SSC (20x):87.7 g of NaCl, 44.1 g of Na citrate add 500 ml of H2O (adjust to pH7.0) Denhards 1 g of Ficoll (type 400), 1 g of (100x):polyvinylpyrrolidones, 1 g of BSA (fraction V) add 50 ml of H2OHybridization 12.5 ml of SSPE (20x), 25 ml of buffer: formamide, 2.5 ofDenhards (100x), 2.5 ml of SDS (10%), 20 mg of tRNA (from brewers yeast,Boeringer Mannheim) RNA sample 10.0 ml of formamide, 3.5 ml of buffer:foraldehyde, 2.0 ml of MOPS (5x) RNA loading 50% of gylcerol, 1 mM ofEDTA, 04% buffer: bromophenol blue

The Northern blot analyses were carried out on the basis of the“Promega” protocol (RNA Applications Guide, Promega Corporation,Madison, USA). 10 μl of RNA preparation were provided with 20 μl ofsample buffer and 5 μl of loading buffer and fractionated on a 1%agarose gel (0.04M MOPS, 0.01M NaAc, 0.001M EDTA, 6.5% formaldehyde, pH7.0). The RNA, gel was blotted by capillary force on a negativelycharged nylon membrane (Boehringer, Mannheim) overnight. The RNA wasfixed on the membrane by UV treatment (1200 kJ for 1 min), and nucleicacids were stained nonspecifically (0.03% methylene blue, 0.3M NaAc).The size standard, and the 18S and 28S RNA were marked and the blot wasdecolorized in water. The membrane was preincubated in 10 ml ofhybridization buffer at 60° C. for 2 h and hybridized overnight afteraddition of a radiolabeled RNA probe. The blot was then evaluated afterstringent washing several times (0.1 SSC, 01% SDS) by exposure on aPhosphor-Imager plate and with the aid of the Molecular Analyst software(Bio-Rad Laboratories, Munich).

Radioactive antisense RNA probes for specific detection of the RNA wereproduced by in vitro transcription using the Riboprobe in vitrotranscription system (Promega, Madison, USA), observing themanufacturer's instructions. The radiolabeled nucleotide used was³²P-α-CTP (10 μCi per reaction). The transcription template employed ineach case was 500 ng of linearized plasmid DNA. An RRE-specific RNAprobe was produced by T7-mediated transcription of XhoI-linearizedpc-ERR. A β-actin-specific RNA probe was produced by SP6-mediatedtranscription of EcoRI-linearized pSP6-actin.

The amounts of cytoplasmic RNA correlated for all the reporterconstructs with the measured GFP expression (FIG. 4, lanes 7-12). In theabsence of the 5′-UTR it was possible to detect only minimal amounts ofhivGFP RNA (hivGFP-RRE) in the nucleus, even when Rev was made availablein trans (FIG. 4, lane 5, 6). In contrast to this, in the presence ofthe authentic 5′-UTR (UTR-hivGFP-RRE), the GFP RNAs adapted to thechoice of codons of HIV structural genes accumulated in the nucleus andwere detectable in large amounts therein (FIG. 4, lane 1).

Without adaptation of the choice of codons to HIV-1 structural genes,the huGFP RNA proved to be stable in the nucleus and was constitutivelytransported into the cytoplasm, even when the 5′-UTR/SD was placed infront of the coding region. It is thus impossible solely by placing anefficiently ultilized SD in front to convert any particular gene intoRev dependence in the absence of an (efficiently utilized) SA.

Rev-dependent GFP expression of hivGFP can be inhibited with therapeuticagents which block Rev-mediated nuclear export. As described in theapplications, it was possible to use the Rev-dependent hivGFP reportersystem described herein for identifying therapeutic agents withantiviral activity, especially those which inhibit the nuclear export ofthe quasi-viral GFP-encoding RNA. Leptomycin B (LMB) and thetransdominant-negative Rev mutant M10 (Rev M10) are the best knowninhibitors of Rev-mediated nuclear export. These established activesubstances should likewise have an inhibitory effect on Rev-dependentexpression of the hivGFP reporter. In order to test this, hivGFPexpression was analyzed in the presence of Rev and LMB or Rev M10.

For the Rev M10 experiments, 3×10⁵ H1299 cells were seeded in a 6-wellcell culture plate and, 24 h later, transfected with 5 μg of reporterplasmid, 2.5 μg of pc-Rev and increasing amounts of pc-RevM10 by calciumphosphate coprecipitation. In addition, the transfection mixture wasadjusted in each case to a total of 15 μg of total DNA with pcDNA 3.1plasmid DNA.

For the LMB experiments, 3×10⁵ H1299 cells were seeded in a 6-well cellculture plate and, 24 h later, transfected with 10 μg of reporterplasmid and 5 μg of pc-Rev or pcDNA 3.1 plasmid DNA by calcium phosphatecoprecipitation. 24 h before harvesting, the medium was supplementedwith 5 nM of LMB. The cells were harvested and GFP expression was readas described above.

It was possible even by cotransfection with equimolar amounts of RevM10to reduce greatly Rev-dependent expression of the UTR-hivGFP-RREreporter construct (FIG. 5A). It was likewise possible to inhibit onlythe expression of the Rev-dependent GFP reporter through the presence of5 nM LMB, but not the expression of the huGFP reporter (FIG. 5B). It wasthus possible to demonstrate that the hivGFP RNA leaves the cell nucleusby the same nuclear export pathway (CRM1) as late HIV-1 transcripts. Itwas additionally possible to demonstrate that GFP expression of theestablished Rev-dependent GFP reporter can be inhibited by Revinhibitors in the same way as the expression of late HIV-1 genes. Thesimple autofluorescent detection of the GFP reporter thus makes thissuitable for identifying Rev inhibitors with antiviral activity.Rev-dependent GFP expression of the hivGFP reporter can be detected on asingle-cell basis and can be quantified by flow cytometry.

The autofluorescent properties of GFP permit detection of Rev-dependentexpression of the hivGFP reporter system on a single-cell basis. Formicroscopic decetion, sterile slides were for this purpose placed inPetri dishes, 10⁶ H1299 cells seeded thereon and and, 24 h later,transfected with 30 μg of GFP reporter plasmid and 15 μg of pc-Rev or 15μg of pcDNA 3.1(+) by calcium phosphate coprecipitation. After 48 h, theslides were washed 2× with PBS, fixed with 4% paraformaldehyde (10 min)and then stained with DAPI (1 mg/ml) at 37° C. for 1 h. Microscopicdetection of the GFP gene product took place with the aid of an OlympusAX500 fluorescent microscope.

In the case of the reporter based on huGFP there was detectable GFPactivity unaffected by the presence and absence of Rev. By contrast,green fluorescent reporter activity of the GFP was detectable inUTR-hivGFP-RRE-transfected cells only when Rev was cotransfected (FIG.6). Detection of the Rev-dependent GFP reporter is thus possible on asingle-cell basis.

In order to be able to quantify this better, in addition the GFPactivity of transfected cells was subjected to an FACS analysis. For theanalysis in a flow cytometer, 10⁶ H1299 cells were seeded in Petridishes and, 24 h later, transfected with 30 μg of reporter construct and15 μg of pc-Rev or pcDNA 3.1(+) by calcium phosphate coprecipitation.The transfected cells were detached by trypsinization 48 h later, takenup in ice-cold PBS and subjected to an FACS analysis. The results havebeen represented in a 2-dimensional diagram. For this purpose, thefluorescence intensity (x axis) was plotted against the cell count (yaxis). It was possible to confirm the results of the expression analysesusing the Western blot technique by the FACS analyses. Once again, theRev/RRE system brought about no increase in GFP expression, irrespectiveof the presence or absence of the 5′-UTR (FIG. 7, 2 to 4). Only in theGFP reporter constructs whose choice of codons was adapted to that ofHIV-1 structural genes and simultaneously had a 5′-UTR/SD was itpossible to detect Rev/RRE-dependent GFP reporter activity. In thepresence of Rev and RRE it was possible to increase the GFP activity ofthe hivGFP reporter from a scarcely detectable background activity (2.2%of huGFP) to more than 15 times the level (36.2% of huGFP). The GFPreporter construct produced on the basis of this invention accordinglypermits quantitative detection of Rev-mediated nuclear export on asingle basis and is thus suitable for high-throughput testing.

utr-1: (SEQ ID NO: 1) gat cga att ccg acg cag gac tcg gct tgc utr-2:(SEQ ID NO: 2) gat ccc atg gct ctc tcc ttc tag cct ccg rre-1: (SEQ IDNO: 3) gat cgg atc cga gat ctt cag acc tgg agg ag rre-2: (SEQ ID NO: 4)gat cct cga ggt tca cta atc gaa tgg atc tg hu-1: (SEQ ID NO: 5) gat cgaatt caa cca tgg tga gca agg gcg agg ag hu-2: (SEQ ID NO: 6) gat cct cgagaa gga tcc ttt act tgt aca gct cgt c cte-1: (SEQ ID NO: 7) gct agg atcccc att atc atc gcc tgg aac cte-2: (SEQ ID NO: 8) cga act cga gca aacaga ggc caa gac atc

TABLE 1 Preferred choice of codons for producing a Rev-dependent gene.Amino acid Codon Priority Ala GCA 1 Ala GCT 2 Arg AGG 2 Arg AGA 1 AsnAAT 1 Asn AAC 2 Asp GAT 2 Asp GAC 1 Cys TGT 1 Cys TGC 2 End TAG 2 EndTAA 1 Gln CAG 2 Gln CAA 1 Glu GAG 2 Glu GAA 1 Gly GGG 2 Gly GGA 1 HisCAT 1 His CAC 2 Ile ATA 1 Ile ATT 2 Leu TTA 1 Leu CTA 2 Lys AAG 2 LysAAA 1 Met ATG 1 Phe TTT 1 Phe TTC 2 Pro CCA 1 Pro CCT 2 Ser AGC 1 SerTCA 2 Thr ACA 1 Thr ACT 2 Trp TGG 1 Tyr TAT 1 Tyr TAC 2 Val GTA 1 ValGTG 2 Priority 1: preferred codon Priority 2: preferred codon if codonof priority 1 is not used.

REFERENCES

-   Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,    Seidman, J. G., Smith, J. A., and Struhl, K. (1994). Percentage of    Codon Synonomous Usage and Frequency of Codon Occurrence in Various    Organisms. Current Protocols in Molecular Biology 2, A1.8-A1.9-   Borg, K. T., Favaro, J. P., and Arrigo, S. J. (1997). Involvement of    human immunodeficiency virus type-1 splice sites in the cytoplasmic    accumulation of viral RNA. Virology 236, 95-103.-   Chang, D. D. and Sharp, P. A. (1989). Regulation by HIV Rev depends    upon recognition of splice sites. Cell 59, 789-795.-   Chen, C. Y. and Shyu, A. B. (1995). AU-rich elements:    characterization and importance in mRNA degradation. Trends.    Biochem. Sci. 20, 465-470.-   Cochrane, A. W., Jones, K. S., Beidas, S., Dillon, P. J., Skalka, A.    M., and Rosen, C. A. (1991). Identification and characterization of    intragenic sequences which repress human immunodeficiency virus    structural gene expression. J. Virol. 65, 5305-5313.-   Graham, F. L. and Eb, A. J. (1973). A new technique for the assay of    infectivity of human adenovirus 5 DNA. Virology 52, 456-467.-   Kaufmann, R. (1979). High level production of proteins in mammalian    cells. Genetic engineering, eds. Setlow, J. K. and Hollaender, A.,    New York, Plenum Press, 155-198.-   Kjems, J., Frankel, A. D., and Sharp, P. A. (1991). Specific    regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev.    Cell 67, 169-178.-   Kjems J. and Sharp, P. A. (1993). The basic domain of Rev from human    immunodeficiency virus type 1 specifically blocks the entry of    U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly. J.    Virol. 67, 4769-4776.-   Kubota, S., Nosaka, T., Furuta, R., Maki, M., Hatanaka, M. (1991).    Functional conversion from HIV-1 Rev to HTLV-1 Rex by mutation.    Biochem. Biophys. Research Commun. 178:1226-1232.-   Laemmli, U. K. (1970). Cleavage of structural proteins during the    assembly of the head of bacteriophage T4. Nature 227, 680-685.-   Lu, X. B., Heimer, J., Rekosh, D., and Hammarskjold, M. L. (1990).    U1 small nuclear RNA plays a direct role in the formation of a    rev-regulated human immunodeficiency virus env mRNA that remains    unspliced. Proc. Natl. Acad. Sci. U.S.A. 87, 7598-7602.-   Luo, Y., Yu, H., and Peterlin, B. M. (1994). Cellular protein    modulates effects of human immunodeficiency virus type 1 Rev. J.    Virol. 68, 3850-3856.-   Maldarelli, F., Martin, M. A., and Strebel, K. (1991).    Identification of posttranscriptionally active inhibitory sequences    in human immunodeficiency virus type 1 RNA: novel level of gene    regulation. J. Virol. 65, 5732-5743.-   Malim, M. H. and Cullen, B. R. (1993). Rev and the fate of pre-mRNA    in the nucleus: implications for the regulation of RNA processing in    eukaryotes. Mol. Cell. Biol. 13, 6180-6189.-   Mikaelian, I., Krieg, M., Gait, M. J., and Karn, J. (1996).    Interactions of INS (CRS) elements and the splicing machinery    regulate the production of Rev-responsive mRNAS. J. Mol. Biol. 257,    246-264.-   Nasioulas, G., Zolotukhin, A. S., Tabernero, C., Solomin, L.,    Cunningham, C. P., Pavlakis, G. N., and Felber, B. K. (1994).    Elements distinct from human immunodeficiency virus type 1 splice    sites are responsible for the Rev dependence of env mRNA. J. Virol.    68, 2986-2993.-   O'Reilly, M. M., McNally, M. T., and Beemon, K. L. (1995). Two    strong 5′ splice sites and competing, suboptimal 3′ splice sites    involved in alternative splicing of human immunodeficiency virus    type 1 RNA. Virology 213, 373-385.-   Olsen, H. S., Cochrane, A. W., and Rosen, C. (1992). Interaction of    cellular factors with intragenic cis-acting repressive sequences    within the HIV genome. Virology 191, 709-715.-   Otero, G. C., Harris, M. E., Donello, J. E. and Hope, T. J. (1998).    Leptomycin B inhibits equine infections anemia virus Rev and feline    immunodeficiency virus Rev function, but not the function of the    hepatitis B virus posttranscriptional regulatory element. J. Virol.    72, 7593-7597.-   Pollard, V. W. and Malim, M. H. (1998). The HIV-1 Rev protein [In    Process Citation]. Annu. Rev. Microbiol. 52:491-532, 491-532.-   Powell, D. M., Amaral, M. C., Wu, J. Y., Maniatis, T., and    Greene, W. C. (1997). HIV Rev-dependent binding of SF2/ASF to the    Rev response element: possible role in Rev-mediated inhibition of    HIV RNA splicing. Proc. Natl. Acad. Sci. U.S.A., 94, 973-978.-   Ratner, L., Fisher, A., Jagodzinski, L. L., Mitsuya, H., Liou, R.    S., Gallo, R. C., and Wong Staal, F. (1987). Complete nucleotide    sequences of functional clones of the AIDS virus. AIDS Res. Hum.    Retroviruses 3, 57-69.-   Rosen, C. A., Terwilliger, E., Dayton, A., Sodroski, J. G., and    Haseltine, W. A. (1988). Intragenic cis-acting art gene-responsive    sequences of the human immunodeficiency virus. Proc. Natl. Acad.    Sci. U.S.A., 85, 2071-2075.-   Schneider, R., Campbell, M., Nasioulas, G., Felber, B. K., and    Pavlakis, G. N. (1997). Inactivation of the human immunodeficiency    virus type 1 inhibitory elements allows Rev-independent expression    of Gag and Gag/protease and particle formation. J. Virol. 71,    4892-4903.-   Schwartz, S., Campbell, M., Nasioulas, G., Harrison, J., Felber, B.    K., and Pavlakis, G. N. (1992a). Mutational inactivation of an    inhibitory sequence in human immunodeficiency virus type 1 results    in Rev-independent gag expression. J. Virol. 66, 7176-7182.-   Schwartz, S., Felber, B. K., and Pavlakis, G. N. (1992b). Distinct    RNA sequences in the gag region of human immunodeficiency virus type    1 decrease RNA stability and inhibit expression in the absence of    Rev protein. J. Virol. 66, 150-159.-   Stauber, R., Gaitanaris, G. A., and Pavlakis, G. N. (1995). Analysis    of trafficking of Rev and transdominant Rev-proteins in living cells    using green fluorescent protein fusions: transdominant Rev blocks    the export of Rev from the nucleus to the cytoplasm. Virology 213,    439-449.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: A sequence comparison of the humanized GFP gene versus thehivGFP gene whose choice of codons has been adapted to that of HIV-1structural genes is depicted. Almost every third wobble position hasbeen replaced mostly by an A or T. This reduces the homology of the tworeading frames to 67.9%. The total AT content thus increases from about37% (huGFP) to about 69% (hivGFP), but without changing the amino acidsequence of the resulting gene products (SEQ ID NOS 9 & 10 are disclosedrespectively in order of appearance).

FIG. 2: Diagrammatic representation of all the GFP reporter constructsproduced and used in this study. Open boxes symbolize GFP genes (hivGFP)adapted to HIV structural genes, and black boxes symbolize GFP genes(huGFP) adapted to mammalian genes. Horizontal lines symbolizeuntranslated regions. All other symbols are explained underneath theimage.

FIG. 3: Expression analysis of the synthetic reading frames. H1299 cellswere transfected with the stated GFP constructs, and cotransfected withthe vectors (+) indicated below the images or blank vector (−). GFPproduction was detected by conventional immunoblot analysis. (A) Testingof a Rev-dependent GFP expression. (B) Testing of GFP expression ofUTR-hivGFP-RRE in the presence of Rev, of a mutated form of Rev (RevM10) and of proviral HIV-1 DNA (HX10). (C) Testing of a CTE-mediated GFPexpression of the reporter constructs. Position and molecular weight ofthe GF protein are indicated by an arrow on the right-hand margin.

FIG. 4: Northern blot analysis and subcellular distribution ofGFP-encoding RNAs. Transfected H1299 cells were partially lysed with0.5% NP-40 buffer, and the nuclei were separated from the cytoplasm bycentrifugation. The total RNA was prepared in each case from the nuclearand cytoplasmic fraction and subjected to a Northern blot analysis. GFPtranscripts adapted to HIV-1 structural genes and adapted to mammaliangenes were detectable simultaneously by means of an RRE-specific probe.The size and position of the GFP RNA is indicated by an arrow. Asinternal control, the RNA of the housekeeping gene β-actin was likewisedetected.

FIG. 5: Effect of Rev inhibitors on the expression of the GFP reporters.H1299 cells were transfected with the stated GFP constructs and analyzedby Western blotting. (A) To test the effect of the transdominant Revmutant (Rev M10) on GFP expression of the UTR-hivGFP-RRE reporter, 5 μgof the reporter were cotransfected with in each case with 2.5 μg of Revplasmid and increasing amounts of Rev M10 expression plasmid (0; 2.5; 5;7.5). (B) To test the effect of LMB on GFP expression, the statedconstructs were cotransfected in the presence (+) and absence (−) of 5nM LMB, and Rev(+) and blank vector (−).

FIG. 6: Immunofluorescence analysis of the huGFP and hivGFP reporter.H1299 cells were transfected with the stated constructs andcotransfected with Rev or blank vector (no Rev) on slides. After 48 h,the cells were fixed and stained with DAPI, and the autofluorescentactivity of the GFP gene product was evaluated in an immunofluorescentmicroscope.

FIG. 7: Flow cytometry analysis of transfected H1299 cells. 1: Mock, 2:huGFP, 3: UTR-huGFP-RRE, 4: UTR-huGFP-RRE and REV., 5: hivGFP-RRE, 6:hivGFP-RRE and REV, 7: UTR-hivGFP-RRE, 8: UTR-hivGFP-RRE and REV. The Yaxis indicates the scattered light intensity, and the X axis indicatesthe GFP-related fluorescence. The vertical line divides the cellpopulations into (left) non-fluorescent and measurable fluorescent GFPactivity (right). The percentage amounts of cells are indicated at thetop in the analyses. The red circles include cell population with highfluorescent GFP activity. The percentage amounts of these cells areindicated below in the analyses (marked red).

FIG. 8: Diagrammatic representation of a Rev-dependent gene. Thesynthetic gene must have a choice of codons which is unusual formammalian genes or a thoroughly high (>50%) A/T content. An untranslatedregion (UTR) which comprises an effective splice donor is positioned 5′from this gene. The viral target sequence must be positioned 3′ fromthis gene. This target sequence may be either a constitutive transportelement such as of the MPMV CTE, or the target sequence of a viral RNAtransport molecule such as of the HIV-1 RRE. Cellular or viral exportfactors then mediate the nuclear export. The Rev-dependent gene is underthe transcriptional control of an inducible (Tet on/off) or constitutive(CMV, SV40) promoter. Polyadenylation of the transcripts is ensured by apolyadenylation signal such as of the BGHpoly(A) signal.

1. A method for detecting RNA export from the cell nucleus of aeukaryotic target cell, comprising (a) providing a polynucleotide whichcodes for a reporter protein and which further comprises (i) acis-active RNA export signal which is in operative linkage with thepolynucleotide (ii) a functional 5′ splice donor without a 3′ spliceacceptor, (iii) an A/T content that is greater than 50%, which reducesthe metabolic stability of a reporter RNA which encodes said reporterprotein in the cell nucleus; (b) introducing the polynucleotide into thecell nucleus of the target cell so that it is present therein inoperative linkage with a transcription control sequence, and that upontranscription of the polynucleotide there is generation of a transcriptin which the segment of the transcript coding for the reporter proteincannot be subjected to any efficient splicing process; (c) providing atrans-active export factor; (d) transcribing the polynucleotide; and (e)determining whether the resulting transcript is exported from the cellnucleus.
 2. The method according to claim 1, wherein the polynucleotideadditionally comprises a choice of codons which is at least partiallyadapted to the choice of codons of exported viral RNA, leading to areduction of the metabolic stability of a reporter RNA in the cellnucleus.
 3. The method according to claim 1, wherein the polynucleotideadditionally comprises a choice of which is at least partially adaptedto the choice of codons of HIV-1 (human immunodeficiency virus 1),leading to a reduction of the metabolic stability of a reporter RNA inthe cell nucleus.
 4. The method according to claim 1, wherein thepolynucleotide which codes for the reporter protein is a syntheticallyproduced polynucleotide.
 5. The method according to claim 1, wherein thereporter protein is a fluorescent protein.
 6. The method according toclaim 5, wherein the fluorescent protein is GFP (green fluorescentprotein), BFP (blue fluorescent protein), RFP (red fluorescent protein),YFP (yellow fluorescent protein), eGFP (enhanced green fluorescentprotein), eBFP (enhanced blue fluorescent protein), eRFP (enhanced redfluorescent protein), eYFP (enhanced yellow fluorescent protein) orhrGFP.
 7. The method according to claim 1, wherein the reporter proteincomprises a detectable enzymatic activity.
 8. The method according toclaim 7, wherein the reporter protein is LUC (luciferase), AP (alkalinephosphatase), SEAP (secretory alkaline phosphatase) or CAT(chloramphenicol acetyltransferase).
 9. The method according to claim 1,wherein the reporter protein is an immunologically detectable protein.10. The method according to claim 1, wherein the reporter protein is aselection marker.
 11. The method according to claim 1, wherein thereporter protein is encoded by a regulatory gene which regulates theexpression of other genes.
 12. The method according to claim 1, whereinthe polynucleotide comprises a cis-active RNA export element.
 13. Themethod according to claim 12, wherein the cis-active RNA export elementis a constitutive RNA export element.
 14. The method according to claim13, wherein the cis-active RNA export element is MPMV CTE (Mason Pfizermonkey virus constitutive transport element), RSV CTE (Rous sarcomavirus constitutive transport element) or SRV CTE (simian retrovirusconstitutive transport element).
 15. The method according to claim 12,wherein the cis-active RNA export element is an export element which isrecognized by a viral export factor.
 16. The method according to claim15, wherein the cis-active RNA export element is HIV-1 RRE (humanimmunodeficiency virus 1 Rev responsive element), HIV-2 RRE (humanimmunodeficiency virus 2 Rev responsive element), SIV RRE (simianimmunodeficiency virus Rev responsive element), HTLV-I (human T-cellleukemia virus I) Rex responsive element or HTLV-II (human T-cellleukemia virus II) Rex responsive element.
 17. The method according toclaim 11, wherein the regulatory gene is a transcription factor.
 18. Themethod according to claim 1, comprising employing a viral cis-active RNAexport element.
 19. The method according to claim 1, wherein the A/Tcontent is increased throughout a reporter construct.
 20. The methodaccording to claim 1, wherein the eukaryotic cell is a mammalian cell.21. A method for detecting RNA export from the cell nucleus of aeukaryotic target cell, comprising (a) providing a polynucleotide whichcodes for a reporter protein and which further comprises (i) acis-active RNA export signal which is in operative linkage with thepolynucleotide (ii) a functional 5′ splice donor without a 3′ spliceacceptor at 3′ from said cis-active RNA export signal (iii) an A/Tcontent that is greater than 50%, which reduces the metabolic stabilityof a reporter RNA which encodes said reporter protein in the cellnucleus; (b) introducing the polynucleotide into the cell nucleus of thetarget cell so that it is present therein in operative linkage with atranscription control sequence, and that upon transcription of thepolynucleotide there is generation of a transcript in which the segmentof the transcript coding for the reporter protein cannot be subjected toany efficient splicing process; (c) providing a trans-active exportfactor; (d) transcribing the polynucleotide; and (e) determining whetherthe resulting transcript is exported from the cell nucleus.